Bacteria need to be cultured in the laboratory for various purposes like identification of the causative agent associated with the pathology, for research purposes, preparations of various vaccines as well as for the productions of certain proteins and enzymes, etc.
The universal culture medium used in the microbiology laboratories that supports the cultivations of almost all types of bacteria is the Trypticase Soy Agar Medium (TSA), also known as the Tryptic soy agar or Tryptone Soya agar. This media provides all the basic components that are required by the bacterial cell for the rapid growth in the laboratory.
The Tryptone Soy Agar (TSA) contains the magical ingredients that supports almost all types of Fastidious i.e. those bacteria that have some specific requirement for the growth as well as the Non-Fastidious bacteria i.e. those bacteria which can grow easily on any medium and have no special requirements, are the Soybean and Casein Digest supported by various components.
However, there are various types of media available that are based on the requirements of particular bacteria but the Trypticase Soy Agar (TSA) is the best artificial medium that fulfills the basic requirements of almost all types of bacteria and gives a satisfactory and rapid growth of most organisms.
In this article, I’m gonna explain the procedure of preparing Trypticase Soy Agar (TSA) medium in Laboratory
PRINCIPLE OF TRYPTICASE SOY AGAR MEDIUM (TSA)
The Trypticase Soy Agar medium (TSA), however not commonly employed in routinely in laboratories, is an excellent medium to obtain the discrete colonies of the bacteria present in the specimen or to get the information about cultural characteristics of bacteria on a solid medium, colony morphology and patterns of growth etc. This Solid medium, used in bacteriology laboratory, constitutes the 6 essential components which are described below in detail –
⇒ Pancreatic digest of casein – The Pancreatic Digest of Casein is a rich source of Nutrients, Vitamins, and Nitrogenous substances that support the rapid growth of the bacterial cell.
⇒ Papaic Digest of Soybean – Similar to Pancreatic Digest of Casein, the Papaic Digest of Soybean is the Rich source of protein or peptides and nutrients that support the rapid growth of the bacterial cell.
⇒ Dextrose – The Dextrose present in the Trypticase Soy Agar (TSA) Medium provides a rich source of carbohydrate for the rapid growth of the Bacterial Cell.
⇒ Dipotassium hydrogen phosphate – It acts as the buffer in the medium that helps to maintain the stable pH of the Trypticase Soy Agar (TSA) medium.
⇒ Sodium Chloride – It maintains the osmotic pressure in the agar medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.
⇒ Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-Anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling 40-45 °C.
The final pH of the solutions is adjusted to 7.1 – 7.5 preferably the 7.3 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the results required so that the rapid and satisfactory growth of the bacterial cell takes place.
The most commonly used modification of TSA medium is the Blood Agar medium containing TSA as a base with added Sheep’s Blood.
The methodology explained in this article is based on HiMedia Labs Tryptic Soya Agar (TSA) Medium which you can check here
REQUIREMENTS FOR PREPARING TRYPTICASE SOY AGAR MEDIUM (TSA)
- Sterile Conical Flask / Erlenmeyer Flask
- Pancreatic digest of casein
- Papaic digest of soybean meal
- Dextrose (Glucose)
- Dipotassium hydrogen phosphate
- Sodium Chloride
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR PREPARING TRYPTICASE SOY AGAR MEDIUM (TSA)
⇒ Weigh the quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, sodium chloride, and Agar-Agar using the weighing scale for 1000 ml of Trypticase Soy Agar medium as follows:
COMPONENTS QUANTITY (in grams)
Pancreatic digest of casein 17.0
Papaic digest of soybean meal 3.0
Dextrose (Glucose) 2.5
Dipotassium hydrogen phosphate 2.5
Sodium Chloride 5.0
Put the butter paper on the weighing scale and transfer the required quantity of Pancreatic digest of casein on to the paper using the spatula. Repeat the step to obtain the required quantity of Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, sodium chloride, and Agar-Agar powder.
Note: Use the separate piece of butter paper to avoid the errors in measurements.
Alternatively, You can use the commercially available Trypticase Soy Agar (TSA) media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 45 grams for 1000 ml medium.
⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.
Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of the solid medium.
⇒ Pour 500 ml of distilled water to the flask and add the weighed quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, and sodium chloride.
⇒ Now add the weighed quantity of Agar-Agar to the above solution.
⇒ Mix well the content and Heat it with continuous agitation to dissolve the constituents.
⇒ Now add more distilled water to the medium and make the volume 1000 ml.
⇒ Check the pH of the solution using pH strip, it should be 7.3 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
⇒ Mix well the content and apply the Non-absorbent cotton plug to the flask.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down to 40-45 °C and pour in the empty media plates under the strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
⇒ Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.
PRECAUTIONS TO BE TAKEN WHILE PREPARING TRYPTICASE SOY AGAR MEDIUM (TSA)
⇒ Carefully measure the quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, sodium chloride, and Agar-Agar as per the quantity of medium to be prepared.
⇒ Maintain the pH of the medium carefully. A single extra drop can change the pH of the solution.
⇒ Store the Medium at low temperature in dust and contamination free environment for later use.
⇒ Acid and Alkali are corrosive to the skin, handle with care.